We have characterized the lipooligosaccharides in three Neisseria meningitidis strains, a wild type and two isogenic variants. Biochemical analyses showed that the wild type expressed three LOS immunotypes, L1, L2, and L3,7. The two isogenic variants had truncated LOSs. One variant expressed L1 and L6 and the other with a deeper truncated LOS was nontypable. Genetic analysis on the LOS biosynthesis genes revealed that the wild type contained four active genes, lgtC,A,B,H, in that order. The two variants had a deletion of one gene, lgtB and the lgtH gene in the deeper truncated LOS variant was inactivated. These caused the changes in phenotypic expressions of LOS in the two variants. The results of biochemical and genetic analyses are consistent. N. polysaccharea is a non-capsulated commensal Neisseria species and was used to classified as non-capsulated N. meningitidis because their similarity in many biochemical properties and morphologies. Using genetic analyses on certain genes in the biosyntheses of LOS, capsular polysaccharide, and outer-membrane proteins as tools, we have reexamined fourteen strains previously classified as N. polysaccharea and reclassified one strain as non-capsulated N. meningitidis. To improve quality control of bacterial vaccine polysaccharides (PSs), we have developed an HPLC method to quantify non-sugar components such as O-acetyl, phosphate, and pyruvate groups in the polysaccharides. The O-acetyl content and the degree of O-acetylation in meningococcal A, C, Y, W-135 PSs, pneumococcal 9V ,18C PSs and typhoid Vi PS were determined. Quantitation of sugar and non-sugar components in polysaccharides (PS) in bacterial PS and PS-conjugate vaccines by HPLC. In the past, we developed an anion-exchange HPLC method to specifically quantify polyribosylribitol phosphate content in vaccines containing Hib conjugates (Vaccine 12:700-706,1994). This HPLC method has been adapted and used by two vaccine manufacturers in quality control of their licensed Hib conjugate vaccines. Because an HPLC method provides higher sensitivity compared to that of a classical colorimetric assay, we have further explored using HPLC methods to quantify sugar and non-sugar components in other bacterial polysaccharide (PS) and PS-conjugate vaccines. Besides monosaccharides, O-acetyl and phosphate are non-sugar components often present in bacterial PSs. The O-acetyl group is an important immunogenic and functional epitope in certain PSs such as meningococcal A and typhoid Vi PSs. In order to insure manufacturing consistency of these and many other PSs and their PS-conjugate vaccines, quality control requires O-acetyl and phosphate contents to be determined and to meet their specifications. Analysis of sugar components in vaccine PSs by HPLC methods has been used in quality control of vaccine manufacturing. Here, we have developed an HPLC method to quantify non-sugar components in bacterial PSs such as O-acetyl, N-acetyl, phosphate, and pyruvate groups. The PSs were treated with sodium hydroxide or acids to hydrolyze these non-sugar components from the PSs. The acetate, phosphate, or pyruvate released in the hydrolysates was then subjected to an anion-exchange HPLC analysis and monitored with a conductivity detector. We have quantified the O-acetyl content of meningococcal A, C, Y, and W-135 PSs, pneumococcal 9V and 18C PSs, and typhoid Vi PS. The HPLC method for the analysis of non-sugar components in vaccine PSs has been accepted for publication. Studies on meningococcal lipooligosaccaride (LOS) and LOS-based conjugate vaccines. Lipooligosaccharide (LOS) is a major surface antigen and a virulence factor of Neisseria meningitidis. Groups B and C meningococci have been the major cause of meningococcal disease in the U.S. Although A, C, Y, W-135 capsular polysaccharides are effective vaccines, the polysaccharide B is ineffective presumably due to its structural similarity to human antigen. Therefore, we have been studying the immunochemistry of Neisseria LOSs in order to develop detoxified LOS-protein complex as a T-dependent immunogen to be a vaccine candidate for group B meningococci. This year, immunochemical and genetic analyses have been done on a meningococcal C wild strain, Tr4, and its two LOS truncated variants, Tr5 and Tr7. The T4 wild-type strain expressed three immunotypes, L1, L2,and L3,7 LOSs. The T5 variant expressed L1 and L6 LOSs due to a truncated LOS. The T7 variant with a deeper truncated LOS was nontypeable. Genetic analysis showed that the gene organization of a locus responsible for the LOS biosynthesis in the T4 wild type was lgtZ,C,A,B,H. The lgtB gene was missing in the two variants and the lgtH gene in the T7 variant was inactivated. The genetic differences in the three strains were consistent with their phenotype changes. The LOS from the T7 variant could be a source material for the LOS-based vaccine since it does not contain an epitope that is present in human tissues. Further investigations are needed to see whether it is a conserved LOS present in N. meningitidis strains including group B organisms. Genetic study of lipooligosaccharides in Neisseria species. We have examined nine lgt genes on three chromosomal loci (lgt-1,2,3) encoding glycosyltransferases responsible for the biosynthesis of LOS in two pathogenic Neisseria, N. meningitidis and N. gonorrhoeae, and 12 species of commensal Neisseria including N. polysaccharea, a nonencapsulated species. The lgt genes in N. polysaccharea were very similar to those in N. meningitidis in composition and organization. The hallmark in distinguishing N. polysaccharea from N. meningitidis is that in the presence of sucrose, the former produces exocellular amylopectin, a starch-like polysaccharide but not a capsular polysaccharide. Fourteen strains of presumably N. polysaccharea all produced amylopectin. However, one particular strain, 93246, reacted with meningococcal monoclonal antibodies to L1 LOS, serotype 4, and serosubtype P1.14. Detailed genetic analysis showed that it contained lgtZ,C.D,E genes as did meningococcal 126E(L1) strain. In addition, it also contained meningococcal opcA gene, and an inactivated SiaD gene, which is a gene in capsular polysaccharide biosynthesis. In consistence with phenotypic expression, strain 93246 contained amS gene that is responsible for the biosynthesis of amylopectin. Therefore, we concluded that strain 93246 is a nonencapsulated N. meningitidis rather than a N. polysaccharea and that it acquired an amS gene through horizontal transfer from a source. To modify the toxicity of LOS for vaccine purpose, meningococcal lpxL2 (htrB2) genes were cloned into the plasmids of E. coli. The construction of lpxL2 mutant in N. meningitidis that alters and detoxify the lipid A is planned. This project incorporates FY2002 projects 1Z01BJ002009-11, 1Z01BJ002010-09, and 1Z01BJ002033-03.